China
Africa
Explore chapters and articles related to this topic
Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
The measurement of proteins using Western blotting involves four essential steps: Protein extraction and denaturation.Separation of proteins by gel electrophoresis.Electrotransfer of proteins from the gel to specific membranes (i.e. nitrocellulose or polyvinylidene difluoride, PVDF).Detection and visualisation of the protein of interest using primary and secondary antibodies.
Biochemical Markers in Ophthalmology
Published in Ching-Yu Cheng, Tien Yin Wong, Ophthalmic Epidemiology, 2022
Abdus Samad Ansari, Pirro G. Hysi
Several conventional low-throughput methods can be utilized in proteomics. Antibody-based methods such as enzyme-linked immunosorbent assay (ELISA) and western blotting depend on the availability of antibodies targeted toward specific proteins or epitopes [142]. Gel-based methods include two-dimensional gel electrophoresis and differential gel electrophoresis. These utilize electric current to separate proteins, before further analysis is completed using alternate testing such as MS [143]. Chromatography-based methods can separate proteins from biological mixtures through various modalities such as ion exchange, size exclusion, and affinity chromatography. Other gel-free chromatographic techniques include gas chromatography and liquid chromatography [144].
Analyzing the GPCR Function of Polycystin-1
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Stephen C. Parnell, Robin L. Maser, Brenda S. Magenheimer, James P. Calvet
Perform Western blotting using anti-HA antibody (Roche Applied Science) and anti-PC1 C-terminal peptide (antibody A19).68 Perform anti-human IgG Western blots as described above and in earlier articles.5,20,68
Overview of gene expression techniques with an emphasis on vitamin D related studies
Published in Current Medical Research and Opinion, 2023
Jeffrey Justin Margret, Sushil K. Jain
Western blotting is used for immunodetection to identify, quantify, and determine the size of proteins. Also referred to as protein- or immune-blotting, this technique is one of the most effective tools used to study proteins. It is accomplished by electrophoretically separating the proteins and transferring them to a membrane for detection using antibodies specific to the target protein11. Once immobilized, a specific antigen is detected and visualized using a primary antibody, secondary antibody peroxidase, and a chemiluminescent reagent12 (Figure 2). This technique was evolved from Southern and Northern blotting and has been used widely for more than three decades. The use of antibody probes against membrane bound proteins has transformed the field of immunology since it has been used to characterize a host of proteins, even those with low abundance13.
Aloperine protects human retinal pigment epithelial cells against hydrogen peroxide–induced oxidative stress and apoptosis through activation of Nrf2/HO-1 pathway
Published in Journal of Receptors and Signal Transduction, 2022
Junhui Zhang, Haitao Zhou, Juanli Chen, Xiaoyan Lv, Hongsong Liu
The whole cell lysates and nuclear protein fractions were prepared, and then, the protein concentrations were determined using Bradford assay (Bio-Rad, California, USA). Samples with equal amounts of proteins were subjected to Western blotting. Proteins were fractionated on 10% SDS-PAGE and then electro-transferred onto a PVDF membrane (Millipore, Billerica, USA). After blocking with 5% skimmed milk in TBST for 1 h, the membranes were incubated overnight at 4 °C with specific primary antibodies for bcl-2 (1:1500), bax (1:1000; Thermo Fisher Scientific, Waltham, MA, USA); Nrf2 (1:500), lamin B1 (1:1000; Abcam, Cambridge, MA, USA), heme oxygenase-1 (HO-1; 1:800) and β-actin (1:1500; Thermo). After that, the membranes were incubated with HRP-labelled secondary antibodies (1:1000; Abcam) for 2 h. Immunoreactivities were visualized with ECL Chemiluminscent Substrate (GE Healthcare, Little Chalfont, UK). The integrated intensities of target bands were determined using Image J software (National Institute of Health, Bethesda, USA).
Polystyrene nanoplastics exposure caused defective neural tube morphogenesis through caveolae-mediated endocytosis and faulty apoptosis
Published in Nanotoxicology, 2021
Jia-hui Nie, Yao Shen, Mohamed Roshdy, Xin Cheng, Guang Wang, Xuesong Yang
Western blotting was performed in accordance with a standard procedure using the antibodies noted in Supplementary Table 1. Protein was isolated from SH-SY5Y cells and tissue homogenates using a radio-immuno-precipitation assay (RIPA, Sigma, MO, USA) buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified using the BCA assay. The extracted protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA). The membrane was blocked with 5% nonfat milk and then incubated with the primary antibody in a TBST buffer at 4 °C overnight. After incubation with the secondary antibody, i.e. HRP goat anti-rabbit IgG or HRP goat anti-mouse IgG, the blots were developed with SuperSignalTM West Femto Chemiluminescent Substrate (ThermoFisher, Rockford, USA) and the Gel Doc™ XR + System (Bio-Rad, CA, USA). Quantity One (Bio-Rad, CA, USA) software was used to capture the chemiluminescent signals and analyze the data.